Suta L.pdf 5 15 [631785]

http://www.revistadechimie.ro REV . CHIM. (Bucharest) ♦ 66 ♦ No. 5 ♦ 2015 718Study Regarding the Behaviour of Encapsulated
Piroxicam in Liquid Media
LENUTA-MARIA SUTA1, IONUT LEDETI2*, LIANA SUCIU3, DANIEL HADARUGA4, ADRIANA FULIAS5, IONELA BELU6
1 University of Medicine and Pharmacy “Victor Babeș”, Faculty of Pharmacy, Department of Pharmaceutical Technology, 2 Eftimie
Murgu Sq., 300041, Timisoara, Romania
2 University of Medicine and Pharmacy “Victor Babeș”, Faculty of Pharmacy, Department of Physical Chemistry, 2 Eftimie Murgu
Sq., 300041, Timisoara, Romania
3 University of Medicine and Pharmacy “Victor Babeș”, F aculty of Pharmacy,Department of Pharmacology and Clinical Pharmacy,
2 Eftimie Murgu Sq., 300041, Timisoara, Romania
4 Politehnica University Timișoara, Faculty of Industrial Chemistry and Environmental Engineering, 6 Carol Telbisz, 300001,
Timișoara, Romania
5 University of Medicine and Pharmacy “Victor Babeș”, F aculty of Pharmacy, Department of Analytical Chemistry, 2 Eftimie Murgu
Sq., 300041, Timisoara, Romania
6 University of Medicine and Pharmacy “Victor Babeș”, Faculty of Pharmacy,Department of Pharmaceutical Technology, 2-4 Petru
Rares Str., 200349, Craiova, Romania
This paper presents the behaviour of piroxicam as active pharmaceutical ingredient vs. piroxicam in or
released from inclusion complexes with α-CD and β-CD, respectively, using reversed phase-high performance/
pressure liquid chromatography (RP-HPLC).
Keywords: piroxicam, inclusion complex, cyclodextrin, HPLC
Inclusion complex (IC) formation between a host
molecule (HM) and a guest molecule (GM) was intensely
studied, especially in the last decades, due to its importancein numerous scientific domains, including pharmaceuticals
[1]. Host molecules are represented by the class of water-
soluble cyclodextrins (CDs), which are cyclic oligo-saccharides consisting in several D-glucopyranose units
that are covalently attached end to end via α-1,4 linkages.
In pharmaceutical domain, the most commonly studiedcyclodextrins are the ones formed by six, seven and eight
glucose units, respectively, which are known as α-CD, β-
CD and γ-CD [2], and due to their structure can form entirely
or partially encapsulation products, with numerous
molecules, including active pharmaceutical ingredients
with low solubility and biodisponibility [3-5].
As mentioned in literature references, the formation of
CDs inclusion complex can be associated with the
presence of intermolecular interactions, such as exclusionof cavity-bounded water, V an der W aals forces, hydrophobic
and/or electrostatic interactions, but as well H-bonding [1,
6]. Several instrumental techniques can be employed asmain investigation tools in evaluation of complex formation
between HM and GM, and the results should be always
corroborated with physico-chemical parameters regardingthe preparation and storage of the formed IC. It is well
known that the formation of the IC depends on parameters
such as ratio between concentration of GM and HM,temperature, experimental protocol for complex formation,
acidity/alcalinity of the medium, the presence of solvent(s)
and co-solvent(s). By the physico-chemical approach, themodification of these parameters leads to the modification
of thermodynamic properties of the system, i.e. to the
modification of the composition during equilibrium [5].
Piroxicam (PX, C
15H13N3O4S, molar mass 331.35 g/mol)
is a non-steroidal anti-inflammatory drug (NSAID)
belonging to a class of oxicams, which is used in treat of
* email: ionut.ledeti@umft.rorheumatoid or osteo-arthritis. The mechanism of action is
not known, but it may block some natural substances,
called prostaglandins, that cause inflammation, treatingonly the pain and inflammation symptoms, not the causes
of those symptoms [7]. The main side effects of PX include
an increased risk of serious digestive complaints likestomach ulcers and bleeding, as well as skin reactions,
insomnia, high blood pressure or kidney and liver damage
[8].
Piroxicam belongs to the Class II drugs according to
Biopharmaceutics Classification System [9], for which the
dissolution is usually the rate-limiting step for
gastrointestinal absorption. To increase the solubility andoral absorption of those drugs from Class II, the formulation
strategies in pharmaceutical field are the synthesis of some
inclusion complexes with different cyclodextrinscontaining the drug as a guest.
This paper presents the results from the research carried
by our group, following some already published papers [2,10, 11], namely the behaviour of pure piroxicam (PX) vs.
piroxicam released from inclusion complexes with α-CD
and β-CD, respectively, using reversed phase-high
performance/pressure liquid chromatography (RP-HPLC).
The structures of the guest molecule (PX) and the ones for
host cyclodextrins ( α-CD and β-CD) are presented in figure
1.
Experimental part
Materials and methods
Piroxicam (PX) with pharmaceutical purity was
obtained as generous gift from LaborMed Pharma,(Romania). Guest molecules ( α-CD and β-CD) were
purchased from (Hungary) and used as received, without
further purification. Solvents for IC preparation (ethanol andbidistilled water) with analytical purity were used, as well
without further purification.

REV . CHIM. (Bucharest) ♦ 66 ♦ No. 5 ♦ 2015 http://www.revistadechimie.ro 719For HPLC analysis, methanol (HPLC grade, Fluka
Chemie AG) and buffer solution of pH=2.5 (citric acid,
Chimopar București and disodium phosphate, Merck& Co.,Inc) were used. The buffer was prepared by mixing 945
mL solution of citric acid 0.1 M and 55 mL solution of
Na
2HPO4 0.2 M.
IC of PX with α-CD and β-CD were prepared according
to a protocol previously established and described by our
group [12], as follows: the selected host molecule, α-CD
and β-CD (62.5 mmol), respectively, was introduced in
bidistilled water (5.0 mL), inside the reaction vessel (heated
minireactor), equipped with controlled monitor oftemperature, condenser, dropping funnel, and placed on a
controllable magnetic stirrer. The minireactor was heated
up to 50
oC, then the equimolar ratio of piroxicam (62.5
mmol) dissolved in ethanol was added dropwise to the
cyclodextrin solution (dropping time 20 min). Following
this, the mixture was kept under heating and stirring foranother 20 min, then slowly cooled to room temperature
and later sealed and refrigerated at +5 °C for 24h. The solid
product that crystallized was filtered under reducedpressure, washed with ethanol and dried at +25 °C. The
yield of complexation was estimated to be 90.2% for α-
CD-IC and 72.6% for β-CD-IC, which are similar to the ones
previously reported [12], proving the reproducibility of the
experimental procedure.The chromatographic analysis RP-HPLC for pure
piroxicam (PX) and the one released from inclusion
complexes α-CD-ICRPX and β-CD-ICRPX was carried out
using a Jasco HPLC system with a PU-2080 Plus Pump,LG-2080-04 Quaternary Gradient unit and DG-2080-54
degasser. The detection was realized using UV-Vis
spectrophotometry (UV-2070 Plus Intelligent UV/VisDetector). The acquired data were processed using the
JASCO ChromPass Chromatography Data System, Version
1.7.403.1. The analysis protocol was set as follows:Nucleosil 100 C18 column with 5 μm particle size;
temperature 25°C, flow 1.0 mL/min, analyzed volume: 20
μL, detection wavelength: 361 nm. As mobile phase, a
mixture of methanol:buffer solution of pH=2.5 in ratio 9:11
was used.
Results and discussions
For the evaluation of controlled release of PX from
encapsulation complexes, the quantitative analysis wascarried out for the compound from solution/suspension of
complex in aqueous or aqueous-ethanol medium, for an
established period of time (min). The quantitativeestimation was achieved using the RP-HPLC protocol
previously described, using a calibration curve Area =
f(concentration) for the pure PX.
Fig. 1. Structure of
guest molecule –
piroxicam (1) and host
cyclodextrins: α-CD
(2) and β-CD (3)
b a
d
c
Fig. 2. The HPLC chromatograms recorded for pure piroxicam (PX) of concentrations (per 100 mL solution): (a): 1.38 mg;
(b): 2.75 mg; (c): 5.5 mg; (d): 11 mg

http://www.revistadechimie.ro REV . CHIM. (Bucharest) ♦ 66 ♦ No. 5 ♦ 2015 720According to this, ethanol solutions (93%) were
prepared, with concentrations of 1.38-22 mg/100 mL for
PX, with a characteristic peak at retention time near 21min (20.5-21.6 min). The chromatograms from HPLC
analysis are presented in figure 2.
The linear correlation Area = f(concentration) is
represented by a coefficient of determination that indicate
a good fitting of the statistical model (r
2=0.99), with the
regression equation:
Area(mV . min) = -2.45 + 14.3871 . c(mg / 100 mL) (fig. 3)
The evaluation of solubilization for the α-CD-PXIC and
β-CD-PXIC and/or controlled release of PX from complexes
was realized by the measuring the concentrations forcomplex or PX in aqueous (for both complexes) or
ethanolic medium (solely for the β-CD-PXIC, which
showed a limited solubility in water).Fig. 2 (cont).The HPLC chromatograms recorded for pure
piroxicam (PX) of concentrations (per 100 mL solution): (e): 22 mge
Fig. 3. The calibration curve for HPLC analysis of PX
The analysis of α -CD-PXIC
The HPLC analysis of aqueous supernatant from the
suspension that contain 0.1 g α-CD-PXIC in 14 mL of
distilled water revealed an increasing of concentration of
bioactive substance (most probably in complex, due tothe decreased retention time comparative to pure PX) in
the first 15 min of stirring, followed by a slight decreasing
after 30 min and later almost constant (fig. 4).
The analysis of
β – CD-PXIC
The considerable lower solubility of β-cyclodextrin and
of the analyzed inclusion complex comparative to the case
of α-cyclodextrin is also sustained by the results obtained
through HPLC analysis. It was evidenced a concentrationup to 10 times lower of complex/bioactive compound in
the supernatant obtained after suspension of 0.1 g β-CD-
PXIC in the same amount of distilled water as previously(14 mL) (fig.5)
Fig. 5. (a) HPLC chromatograms for supernatant samples from ethanol (93%) suspensions of β-CD-PXIC obtained on 0-60 min
time interval; (b) the variation of concentration for β-CD-PXIC in supernatant vs. timeFig. 4. (a) HPLC chromatograms for supernatant samples from aqueous suspensions of α-CD-PXIC obtained on 0-75 min
time interval; (b) the variation of concentration for α-CD-PXIC in supernatant vs. time
(a) (b)

REV . CHIM. (Bucharest) ♦ 66 ♦ No. 5 ♦ 2015 http://www.revistadechimie.ro 721When using the solubilization/controlled release in
ethanol 93%, for the β-CD-PXIC, a maximum concentration
was observed after one minute of stirring, followed by a
drastically decreasing of concentration for bioactive
compound beyond half of initial value (fig.6.). Also, it wasnoticed that two chromatographic peaks appear at close
retention time values, which can be associated with the
appearance of another encapsulation complex, or to partialdissociation of the complex in the presence of ethanol.
Conclusions
This study presented the behaviour in condensed phase
(in two polar solvents) of two inclusion complexes of α-
cyclodextrin and β-cyclodextrin with piroxicam, which is
a non-steroidal anti-inflammatory with low water-solubility.
For the evaluation of controlled release of piroxicam
from encapsulation complexes, the quantitative analysiswas carried out for the compound from solution/
suspension of complex in aqueous or aqueous-ethanol
medium. The quantitative estimation was achieved usingthe RP-HPLC protocol.References
1. YANG H., HILLIER S.L., PARNIAK M.A., ROHAN L.C., J. Incl. Phenom.
Macrocycl. Chem., 72, 2012, p. 459.2. ăUTA L.M., VLAIA L., FULIAS A., LEDETI I, HADARUGA D., MIRCIOIU
C., Rev. Chim. (Bucharest), 64, no. 11, 2013, p. 1279.
3. CHALLA R., AHUJA A., ALI JAVED, KHAR R.K., AAPS Pharm. Sci.Tech. 6(2), 2005, p. 329.
4. MOSHER G., THOMPSON D.O., Complexation: Cyclodextrins în
Swarbrick J. Encyclopedia of Pharmaceutical Technology, 3rd ed.,vol. 2, Informa Healthcare, New York, 2007, p. 671-696.
5. LOFTSSON T., BREWSTER M.E., J. Pharm. Sci. 85(10), 1996, p. 1017.
6. LIU L., GUO Q.-X., J. Incl. Phenom. Macrocycl. Chem. 42, 2002, p. 1.
7. Royal Pharmaceutical Society of Great Britain, British National
Formulary, British Medical Association: London, UK, 2004; p 476.
8. SCARPIGNATO C., Curr. Med. Chem., 20(19), 2013, p. 2415.9. AMIDON G.L.. LENNERNAS H., SHAH V .P ., CRISON J.R., Pharm. Res.
12, 1995, p. 413.
10.
SUTA L.M., VLAIA L., VLAIA V ., OLARIU I., HADARUGA D. I., MIRCIOIU
C., Farmacia, 4(60), 2012, p. 475.
11. MICLEA L.M., VLAIA L., VLAIA V ., HADARUGA D. I., MIRCIOIU C.,
Farmacia, 5(58), 2010, p. 583.12. HADARUGA D.I., HADARUGA N.G., MICLEA L.M., VLAIA L.,
MIRCIOIU C., J. Agroalim. Proc. Tech., 2009, 15(4) , p.478
Manuscript received: 25.08.2014
Fig. 6. (a) HPLC chromatograms for supernatant samples from ethanol (93%) suspensions of β-CD-PXIC obtained
on 0-45 min time interval; (b) the variation of concentration for β-CD-PXIC in supernatant vs. time